A broad spectrum of human diseases ("laminopathies") is caused by mutations in components of the nuclear lamina, a protein meshwork that lines the nuclear envelope. Many of these disease mutations affect heart and skeletal muscle. The prototype laminopathy is Emery-Dreifuss muscular dystrophy (EDMD), which causes dilated cardiomyopathy with conduction defects (DCM-CD) and dystrophy of skeletal muscles, and is caused by mutations in either lamin A or in inner nuclear membrane (INM) protein emerin. While the molecular basis for laminopathies is unknown, proximal causes are speculated to involve defects in cell signaling. EDMD models show elevated ERK1/2 signaling in heart, a pathway known to be linked to pathological cardiac hypertrophy. The broad, long-term objectives of this work are to understand how components of the nuclear lamina regulate signaling and contribute to normal and pathophysiological processes in heart and skeletal muscle. The current project is aimed at analyzing two INM proteins associated with the nuclear lamina that were recently shown to regulate signaling in myoblast differentiation, Lem2 and Net37. Lem2 (gene name, Lemd2) is involved in attenuation of ERK1/2 signaling, and Net37 is needed for secretion of IGF-II, a factor essential for autocrine signaling in myogenesis. The work will use cultured myoblasts to analyze how these INM proteins control signaling at the molecular level, and mouse models to understand their functions in heart and skeletal muscle and their potential relevance to diseases. Aim 1 will dissect the regions of Lem2 required for ERK attenuation, identify potential ERK regulators that interact with these regions by proteomics, and functionally analyze these components in myoblast differentiation. Aim 2 will evaluate whether Lemd2 can functionally compensate for loss of Emd in mouse, by analyzing animals with a hemizygous Lemd2 gene trapped allele in an emerin-null background. Also, a floxed allele of Lemd2 will be constructed for tissue-specific knockouts in adult heart and skeletal muscle, to evaluate the importance of Lemd2 in cardiac maintenance and in skeletal muscle function and repair. Aim 3 will dissect the regions of Net37 required for IGF-II secretion and myoblast differentiation, analyze how the putative glycosidase activity of its luminal domain is involved in IGF-II folding and secretion, and determine whether Net37 and IGF-II have distinctive functions in muscle regeneration in mouse after silencing the endogenous genes by AAV vectors. It also will investigate whether the binding of signaling factors to the nucleoplasmic side of Net37 regulates the activity of its luminal domain. Overall this project is expected to shed significant new light on the molecular functions of Lem2 and Net37, and the results are predicted to provide insight on their functions in normal heart and skeletal muscle and in pathophysiological processes in these tissues. PUBLIC HEALTH RELEVANCE: This work will analyze proteins involved in controlling molecular pathways that are of fundamental importance to heart and skeletal muscle. The studies are expected to enhance an understanding of how genetic mutations in these proteins may cause certain cardiac diseases and skeletal muscle dystrophies, and could contribute to improvement of therapeutic options for treatment of these diseases.